The availability of T-cells mutants that lack one or more of the T-cell antigen receptor chains, and clones of the genes that encode these chains, has allowed us to reconstruct a cell surface TCR that expresses well defined alterations. These studies have shown that the Zeta chain, which has a longer intracytoplasmic portion than any of the CD3 subunits, is critical in allowing TCR occupancy to result in IL-2 secretion. In contrast, the role of CD3-delta appears to be largely confined to assembly and transport of the TCR to the cell surface. The introduction of an active tyrosine kinase (encoded by v-src) into T cells allowed the study this signalling pathway in isolation. Although causing spontaneous IL-2 production, there was no evidence that the PI hydrolysis pathway or protein kinase C are involved. Surprisingly, intracellular Ca2+ levels were higher, and TCR-mediated Ca2+ increases were markedly enhanced in the v-src+ cells, suggesting the novel hypothesis that tyrosine kinases may directly regulate [Ca2+]i in T cells. In addition, the completely normal responses (PI hydrolysis, CD3-Gamma phosphorylation, growth inhibition, programmed cell death, IL-2 production) of these cells suggests that the theory that phosphorylation of TCR Zeta desensitizes this receptor is, at best, an oversimplification. Finally, heterochimeric antibodies (made of 2 F(ab) fragments, one against the TCR and the other against an MHC class II molecule) were used to stimulate T cells with mutated TCR Zeta chains. When bound to plastic, the antibodies stimulated these mutants and the wild type equally, but when "presented" by an accessory cell only the wild type responded. This is identical to the phenotype of intact, plastic-bound antibody vs. antigen. These data will be used to develop a model of how TCR occupancy initiates T-cell activation.